ABSTRACT: In the United States gross mortality from invasive fungal disease is approximately 50%. People with AIDS, chemotherapy patients, and transplant recipients are at highest risk of acquiring life-threatening infections, but many fungi also cause disease in apparently healthy individuals. The environmental yeast Cryptococcus epitomizes this trend. Like many fungi, infection with Cryptococcus occurs when it is inhaled into the lung from which it can disseminate to the central nervous system (CNS) and cause disease. Once in the CNS Cryptococcus causes fungal meningoencephalitis that is fatal ~25% of the time, even with state-of-the-art treatment. This high incidence of mortality is consistent with other invasive fungal diseases and indicative of the dire need for improved therapeutics. To develop new strategies for combating invasive fungal pathogens, it is imperative that we gain a better understanding of the fundamental biology of fungal pathogens. Our long- term research goal is to understand the properties of fungal infectious particles and how they cause disease. In Cryptococcus, both yeast and spores are likely infectious particles of humans, but studies of the pathogenic potential of spores were historically hampered by technical limitations. Recently, however, we purified spores to homogeneity in numbers sufficient for comprehensive biochemical, molecular, and virulence studies. Using this novel reagent, we discovered that spores can cause disease in a mouse model, providing the first evidence that spores can act as infectious particles in mammalian cryptococcosis. The objective of this proposed project is to investigate for the first time the key processes by which fungal spores transition from quiescent cells into vegetatively growing yeast (germinate) and infect the mammalian lung. Our central hypothesis is that by determining the cellular and molecular mechanisms governing spore germination and lung invasion, we will identify key events in spore-mediated infections that can be targeted for inhibition. To test this hypothesis, we will carry out two Specific Aims: 1) identify the cellular and molecular events that occur during germination of spores and 2) determine the mechanism(s) by which spores cross the epithelium to colonize the lung. We will combine molecular and classical genetics, gene expression data, protein composition data, and quantitative high-resolution germination assays to parse the germination process into discrete events. At the same time we will use in vitro tissue culture models, a new organotypic microlung model, and a mouse intranasal model of infection to determine how spores colonize the lung. These innovative experiments will result in an in-depth map of germination pathways and insights into how spores invade the host. Understanding pathways and processes associated with the two pivotal events that occur during spore- mediated disease (germination and infection) makes significant contributions to the long-term objective of this work to identify new and diverse molecular, cellular, and systemic targets that can be exploited for novel antifungal therapeutic strategies to prevent and/or treat cryptococcosis and other fatal human fungal diseases.